Fig. 4.
Phosphorylation of Tip60-T158 by p38 induces the acetyltransferase activity of Tip60 and is required for the ability of Tip60 to mediate senescence.
(A) Increasing amounts of Tip60α or buffer (−) was incubated first with p38α and MKK6E with (+) or without (−) ATP and then with histone and acetyl-CoA.
(B) Increasing amounts of wild type or indicated mutant of Tip60α or buffer (−) was incubated first with p38α, MKK6E and SB203580 (SB) or vehicle control (Ctrl) and then with histone and acetyl-CoA.
(C) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with FLAG-Tip60α (WT) or -Tip60α-A158 or vector (Ctrl) and HaRasV12 (Ras), MKK3E (K3E) or vector (VT), and incubated with histone and acetyl-CoA.
(D) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with FLAG-Tip60α; shRNA for GFP or p38α (756, 785) or p38δ (386, 695); and HaRasV12 (Ras) or vector (VT), and incubated with histone and acetyl-CoA.
(A–D) Acetylated histone H4 was detected by Western blotting using an anti-acetyl-Lys antibody. Substrate input and recombinant proteins were stained with Ponceau. Part of the IPs and lysates were analyzed by Western blotting (C, D).
(E) FLAG-Tip60 was immunoprecipitated from BJ cells transduced with FLAG-Tip60α (WT), -Tip60α-D98, -Tip60α-D158, -Tip60β (WT) or -Tip60β-D106 and incubated with histone and [14C]acetyl-CoA. Reactions were resolved by SDS-PAGE. Acetylation of histone H4 was detected by autoradiography. Histone input was stained with Coomassie Brilliant Blue. Part of the IPs were analyzed by Western blotting.
(F) Population doublings of BJ cells transduced with shRNA for GFP or Tip60 (sh887); mTip60 (left panel), mTip60-A155 (middle panel), mTip60-A158 (right panel) or vector (WN); and HaRasV12 (Ras) or vector (WH) over 10 days, starting on day-4 post ras transduction.
(G) % of SA-β-gal positive cells in cell populations described in (F).
(F–G) Values are mean ± SD for triplicates.
See also Figure S4.